The heart's pumping capacity is determined by myofilament power generation. Power is work done per unit time and measured as the product of force and velocity. At a sarcomere level, these contractile properties are linked to the number of attached cross-bridges and their cycling rate, and many signaling pathways modulate one or both factors. We previously showed that power is increased in rodent permeabilized cardiac myocytes following PKA-mediated phosphorylation of myofibrillar proteins. The current study found that that PKA increased power by ∼30% in permeabilized cardiac myocyte preparations (n = 8) from human failing hearts. To address myofilament molecular specificity of PKA effects, mechanical properties were measured in rat permeabilized slow-twitch skeletal muscle fibers before and after exchange of endogenous slow skeletal troponin with recombinant human Tn complex that contains cardiac (c)TnT, cTnC and either wildtype (WT) cTnI or pseudo-phosphorylated cTnI at sites Ser23/24Asp, Tyr26Glu, or the combinatorial Ser23/24Asp and Tyr26Glu. We found that cTnI Ser23/24Asp, Tyr26Glu, and combinatorial Ser23/24Asp and Tyr26Glu were sufficient to increase power by ∼20%. Next, we determined whether pseudo-phosphorylated cTnI at Ser23/24 was sufficient to increase power in cardiac myocytes from human failing hearts. Following cTn exchange that included cTnI Ser23/24Asp, power output increased ∼20% in permeabilized cardiac myocyte preparations (n = 6) from the left ventricle of human failing hearts. These results implicate cTnI N-terminal phosphorylation as a molecular regulator of myocyte power and could serve as a regional target for small molecule therapy to unmask myocyte power reserve capacity in human failing hearts.